TY - JOUR
T1 - Development of an immobilized P-glycoprotein stationary phase for on-line liquid chromatographic determination of drug-binding affinities
AU - Zhang, Yanxiao
AU - Leonessa, Fabio
AU - Clarke, Robert
AU - Wainer, Irving W.
N1 - Funding Information:
This work was supported by NIH grant 1R426M56591-02 (IWW) and USAMRMCRP9550649 (RC).
PY - 2000/2/28
Y1 - 2000/2/28
N2 - The membrane transporter P-glycoprotein (PGP) has been immobilized on an immobilized artificial membrane (IAM) LC stationary phase. The resulting PGP-IAM phase retained the ability of the native PGP to bind the known PGP-ligand vinblastine. Displacement studies using other known PGP ligands, verapamil and cyclosporin A, demonstrated that there was selective binding between vinblastine and the immobilized PGP transporter. The binding affinity (K(d) value) of vinblastine for the PGP-IAM was determined to be 19±20 and 71±11 nM on two separate columns. These values are consistent with previously reported values of 9±2, 8±2, and 37±10 nM, which were obtained using native membranes. The K(d) values obtained on the PGP-IAM for cyclosporin A and verapamil were 492±21 and 172±29 μM, respectively. These results were higher than the corresponding K(d) values obtained using native membranes, but the relative affinities vinblastine>cyclosporin A>>verapamil are consistent in both approaches. During several months of experiments and storage, the PGP-IAM was found to be reproducible and stable. The stationary phase appears to be useful in the on-line screening for PGP ligands. Copyright (C) 2000 Elsevier Science B.V.
AB - The membrane transporter P-glycoprotein (PGP) has been immobilized on an immobilized artificial membrane (IAM) LC stationary phase. The resulting PGP-IAM phase retained the ability of the native PGP to bind the known PGP-ligand vinblastine. Displacement studies using other known PGP ligands, verapamil and cyclosporin A, demonstrated that there was selective binding between vinblastine and the immobilized PGP transporter. The binding affinity (K(d) value) of vinblastine for the PGP-IAM was determined to be 19±20 and 71±11 nM on two separate columns. These values are consistent with previously reported values of 9±2, 8±2, and 37±10 nM, which were obtained using native membranes. The K(d) values obtained on the PGP-IAM for cyclosporin A and verapamil were 492±21 and 172±29 μM, respectively. These results were higher than the corresponding K(d) values obtained using native membranes, but the relative affinities vinblastine>cyclosporin A>>verapamil are consistent in both approaches. During several months of experiments and storage, the PGP-IAM was found to be reproducible and stable. The stationary phase appears to be useful in the on-line screening for PGP ligands. Copyright (C) 2000 Elsevier Science B.V.
KW - Cyclosporin A
KW - Drug-binding affinities
KW - P-Glycoprotein
KW - Stationary phases, LC
KW - Verapamil
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U2 - 10.1016/S0378-4347(99)00384-9
DO - 10.1016/S0378-4347(99)00384-9
M3 - Article
C2 - 10744311
AN - SCOPUS:0033970167
SN - 1387-2273
VL - 739
SP - 33
EP - 37
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 1
ER -